Analysis and detection of pathogens in food and other types of samples involves multiple aspects. One aspect often involves growing and enriching a food sample to increase the number of target micro-organisms in the sample up to detectable (or more readily detectable) levels. For example, U.S. Pat. No. 6,312,930; WO 98120148; U.S. Pat. No. 5,843,669; U.S. Pat. No. 5,145,786; and EP 1 253 203 published Oct. 30, 2002, discuss enrichment and pre-enrichment of such samples.
Another aspect of analysis and detection of pathogens in food and other types of samples relates to isolation or collection of the cells in the sample so that such cells can be conveniently further processed, which processing may include disruption of the cells if the analysis relates to intracellular components of the cells. For example, if the sample aliquot contains yeast, fungal cells or spores, and it is desired to analyze the nucleic acid contained within the cells or spores, the cells or spores are optionally enriched, and then must be collected and disrupted to release their intracellular contents, including the nucleic acids. The process of collecting fungal cells prior to analysis often involves filtration, using for example vacuum filtration. The collected filtration retentate, which comprises the cells and spores of the fungal cells, must then traditionally be removed from the surface of the filtration medium, which typically comprises a filter paper, prior to cellular disruption.
One method of cell disruption involves subjecting cells or spores to forces which mechanically disrupt the cell walls, cell membrane, and other component structures of the cells or the spores, to release the internal cell contents into solution. For example, in a technique referred to in the art as “bead beating,” as described in references WO 98/11257 and WO 2003008636, a sample solution comprising cells or spores is introduced into a container having physically disrupting elements. The sample solution is agitated to cause disruptive contact between the cells or spores and the disrupting elements, sufficient to disrupt the cells or spores and release their contents. Such mechanical disruption can optionally be followed by chemical or enzymatic lysis to complete the disruption or further digest the remaining cellular debris.
In a typical prior art method, a sample solution, which optionally has been previously enriched and filtered, can be introduced into a test tube containing disruption beads. The combined solution containing the target micro-organisms and the beads is then subjected to an application of force (for example, centrifuge, vortex, etc.), which physically disrupts the cells. The solution containing the disrupted cellular content, for example the released nucleic acids, is then suitable for analysis, including PCR-based detection analysis.
In some prior art methods involving algae, disruption of filtered cells is carried out without first removing the filter retentate from the filtration medium, i.e. disrupting the entire filter and the accompanying retentate. For example, methods involving cell disruption of algae on a filtration medium without first removing the retentate from the filtration medium are described in Schumann et al., Chlorophyll extraction methods for the quantification of green microalgae colonizing building facades, International Biodeterioration & Biodegradation, vol. 55, pp. 213-22 (2005); Rossi et al., Lipid biomarkers and trophic linkages between phytoplankton, zooplankton and anchovy (Engraulis encasicolus) larvae in the NW Mediterranean, Journal of Plankton Research, vol. 28, pp. 551-62 (2006); and Eikrem et al., Florenciella parvula gen. et ap. Nov. (Dictyochophycae, Heterokontophyta), a small flagellate isolated from the English Channel, Phycologia, vol. 43, pp. 658-68 (2004). In these prior art methods, the algae are filtered onto glass fiber filters (e.g., Whatman GF/F filters) and then the filters are placed into a physical disruption vessel (e.g., a Vibrogen IV bead mill) where the filter and accompanying cells are disrupted by bead beating. It should be noted that, in the cited references involving disruption of algae without prior removal of retentate from the filtration medium, the technique is employed specifically to release and characterize chlorophyll and pigments for further analysis, and not to release and examine nucleic acids.
An additional aspect of analysis and detection of pathogens in food and other types of samples relates to the actual method of detection utilized after the cells have been obtained. Frequently, detection methods are used which employ the polymerase chain reaction (PCR) method of nucleic acid amplification. One such detection method involves melting curve analysis of PCR amplification products, which method is described in detail in PCT Publication Nos. WO 97/11197 and WO 00/66777 and U.S. Pat. No. 6,312,930.